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Isolation of a Complementary DNA
Clone for the Human Complement
Protein C2 and Its Use
in the Identification of a Restriction
Fragment Length Polymorphism
Derek E. Woods, Michael D. Edge, and Harvey R. Colten
Division of Cell Biology, Department ofMedicine, Children's
Hospital, Ina Sue Perlmutter Cystic Fibrosis Research Center,
Department ofPediatrics, Harvard Medical School, Boston,
Massachusetts 02115; Imperial Chemical Industries,
Pharmaceuticals Division, Mereside, Alderly Park,
Macclesfield, Chesire, England
Abstract. Complementary DNA (cDNA) clones
corresponding to the major histocompatibility (MHC)
class III antigen, complement protein C2, have been isolated from human liver cDNA libraries with the use of
a complex mixture of synthetic oligonucleotides (17 mer)
that contains 576 different oligonucleotide sequences. The
C2 cDNA were used to identify a DNA restriction enzyme
fragment length polymorphism that provides a genetic
marker within the MHC that was not detectable at the
protein level. An extensive search for genomic polymorphisms using a cDNA clone for another MHC class III
gene, factor B, failed to reveal any DNA variants. The
genomic variants detected with the C2 cDNA probe provide an additional genetic marker for analysis of MHClinked diseases.
Introduction
The genes for three of the serum complement proteins, the
second (C2) and fourth (C4) components and factor B, have
been localized to the major histocompatibility complex (MHC)
in humans, mice, and guinea pigs (1) and have been designated
class III MHC antigens. Human C4 is a three-chain glycoprotein
200,000 Mr coded for by two loci, C4A and C4B on the short
arm of chromosome 6 (2). The products of these loci differ
structurally (3) and functionally (4) and several protein polymorphic variants have been identified for each locus (4). Factors
B and C2 are single chain glycoproteins of95,000 (5) and 102,000
-
Received for publication 20 January 1984 and in revised form S April
1984.
J. Clin. Invest.
© The American Society for Clinical Investigation, Inc.
0021-9738/84/08/0634/05 $ 1.00
Volume 74, August 1984, 634-638
634
D. E. Woods, M. D. Edge, and H. R. Colten
Mr (6), respectively, which function in the alternative and classical pathways of complement activation. Several genetic variants
of factor B protein have been identified by differences in mobility
on agarose electrophoresis. These have been useful in analysis
of populations and families with MHC-linked disorders such
as insulin dependent juvenile onset diabetes mellitus (7).
C2 protein variants have been recognized by isoelectric focusing of serum samples (8) in polyacrylamide gels and detected
functionally by overlaying with an agarose gel containing antibody-sensitized sheep erythrocytes and either C2-deficient human serum or dilute normal human serum. These reveal a
common variant, C2C, a basic variant in 4-5% of unrelated
individuals designed C2B, and rarely an acidic variant (1). Two
such acidic C2 variants, designated C2 Al and C2 A2 have
been recognized in <1% of the population. Individually, the
class III gene products display less polymorphism than the class
I and class II proteins.
Recently, cDNA clones corresponding to human and mouse
class I and II histocompatibility antigens (HLA and H2) (9-13)
and the class III proteins factor B (14, 15) and C4 (16-18) have
been isolated and characterized. The availability of cDNA probes
for genes within the MHC have permitted an analysis of the
fine structure of this region (13, 19). Additional structural data
derived from nucleotide sequencing will lead to an understanding
of the molecular basis of polymorphic variants important in
regulation of the immune response.
DNA polymorphisms of the C4 genes have been identified
by two groups (20, 21). These C4 DNA polymorphisms were
detected among individuals who were homozygous as defined
by C4 protein variants. This permitted subdivision of the C4
variants already identified and therefore generated additional
markers within the MHC. These results prompted efforts to
isolate human C2 cDNA clones that could be used together
with factor B cDNA (14) probes to generate additional markers
for analysis of MHC-linked diseases. Genomic polymorphisms
at a given locus are detected by digestion of DNA samples with
restriction endonucleases that recognize specific nucleotide se-
Downloaded from http://www.jci.org on June 15, 2017. https://doi.org/10.1172/JCI111461
quences. The resulting DNA fragments are size-separated electrophoretically and transferred to nitrocellulose filters. Radiolabeled DNA probes are then used to detect fragments that
contain complementary sequences (22).
In this report, we describe the isolation of cDNA clones for
human C2. The C2 clone and a factor B clone (14) have been
used to investigate polymorphisms of these genes at the DNA
level.
Methods
Isolation of C2 cDNA clones. A 17-nucleotide long oligonucleotide mixture (Fig. 1) was synthesized by a solid-phase phosphotriester method
(14). In position of nucleotide 7 and 13 for the glutamine (Gln) codons,
25% cytosine (C) was added as well as the guanosine (G) in order to
obtain representation of the codon for glutamic acid (Glu). The oligonucleotide mixture was kinase labeled with [32P]dATP (23) and
used to screen a human liver cDNA library, as described by Woods et
al. (14).
Approximately 40,000 clones were screened. The final posthybridization washes were at 45°C in 6 X SSC (0.15 M NaCl, 0.015 M
Na citrate, and 0.05% sodium pyrophosphate).' Positive clones were
colony purified and rescreened as described above. The five positive
clones isolated were used as probes in Northern blot analysis (24) against
human liver mRNA to determine if they hybridized to a mRNA of
approximately the correct size. One of the clones, pC2-10 (-500 base
pairs (bp)), was partially sequenced by using the Maxam and Gilbert
sequencing procedure (23). pC2-10 was used to rescreen 200,000 clones
of the human adult liver cDNA library ( 14) and 15 additional C2 clones
were identified. The largest of these was pC2-6 (900 bp). This insert was
used to screen a human fetal liver cDNA library (25) and a C2 clone
(pC2-F2) of 1.6 kilobases (kb) was isolated.
Restriction enzyme analysis of human DNA. Restriction enzyme
digestions were carried out and DNA samples were analyzed by the
Southern blot technique (22). The insert from the C2 clone (pC2-6) and
the factor B clone (pBfA28) (14) were nick translated (26) and used as
probes.
Complement protein typing. Blood for genetic typing of BF, C2, and
C4 was collected into 1 mg/ml of sodium ethylenediaminetetra-acetate
(Na2 EDTA). Plasma was obtained by centrifugation and immediately
frozen and stored at -80°C. The specimens were thawed immediately
before analysis.
Plasma samples were subjected to agarose gel electrophoresis. Factor
B patterns were visualized by immunofixation with goat anti-human B
(Atlantic Antibodies, Scarborough, ME) as described previously (27).
For C2 typing, plasma samples were subjected to isoelectric focusing in
polyacrylamide gels (8) and C2 patterns were developed with an agarose
gel overlay containing antibody-sensitized sheep erythrocytes and fresh
normal human serum diluted 1:90 instead of C2-deficient human serum
as originally described.
For C4 typing, plasma samples were desialated and detected by
electrophoresis ofdesialated plasma in agarose gels and then immunofixed
with goat anti-human C4 antiserum (Atlantic Antibodies) (4).
Complement genetic nomenclature. The nomenclature for genetic
polymorphism of the complement proteins conforms to the International
1. Abbreviations used in this paper: SSC, 0.15 M NaCl, 0.015 M Na
citrate, and 0.05% sodium pyrophosphate.
System for Human Gene Nomenclature (1979) (28) as revised at the
4th International Workshop for the Genetics of Complement, Boston,
July 1982. Alleles of the complotypes are written in the order BF, C2,
C4A, and C4B although the true order of the genes is C2, BF, C4A,
and C4B (29).
Results
The C2 oligonucleotide mixture shown in Fig. 1 was kinase
labeled and used as a probe to screen 40,000 clones of the
human adult liver cDNA library. The nitrocellulose filters were
washed in 6 X SSC at 45°C and autoradiographed. Under these
conditions, five weakly hybridizing clones were identified. Analysis ofthese clones by hybridization to a Northern blot of human
liver mRNA showed that only two clones hybridized to mRNA
large enough to code for C2 i.e., -2.7 kb. One of these clones,
pC2-10 (-500 bp), was partially sequenced by using Maxam
and Gilbert DNA sequencing; the results are shown in Fig. 2.
This sequence represents the 5'-terminus of the clone, which
corresponds to the amino-terminus of the C2a fragment (30)
(the 70,000-D carboxyterminal fragment of C2 generated by
the Cl enzyme during C2 activation) plus the nucleotides for
five amino acids of the carboxyterminus of the C2b fragment.
Fig. 2 also shows a comparison ofthe protein sequence generated
from the cDNA sequence (a), the published sequence for the
amino-terminus of human C2a (30) (b), and the published sequence of guinea pig C2a (30) (c). The amino acid sequence
derived from the cDNA sequence is homologous to the published
amino acid sequences for both human and guinea pig C2, thus
identifying pC2-10 as a C2 cDNA clone. The amino acid sequence derived from the cDNA clone differs from the published
human C2a protein sequence at residue 6 (Arg for Ser) and 10
(Leu for Lys). Differences between the derived human C2a sequences and the published sequence for guinea pig C2a (30)
were evident at positions 18, 20, 24, 25, 27 and 28 (Fig. 2).
DNA isolated from leukocytes of six unrelated individuals
(Glu)
(Glu)
Protein Sequence (30)
Lys Ile Gln Ile Gln Ser
1
2
3
4
5
6
AAG AUA CAA AUA CAA UC
A
C(G)G
C(G)G AG
U
U
TTC TAT GTT TAT GTT AG
T
G(C)C
G(C)C TC
A
A
I
mRNA Sequence
Oligonucleotide Sequence
Figure 1. Amino acid sequence of human C2a used to construct the
synthetic oligonucleotide mixture, which was then used to isolate C2
cDNA clones. The derived potential mRNA sequences and the oligonucleotide sequence are shown.
635
Human Complement (C2) DNA Polymorphism
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Figure 2. Partial nucleotide sequence of human C2 cDNA
clone pC2-10 and amino acid
sequence derived from the nucleotide sequence (a); published amino acid sequence of
the amino terminal segment of
human C2a (b); and guinea pig
C2a (30) (c). Amino acid residues shown in bold face indicate position of the sequence
used to construct the oligonucleotide mixture. Residues underlined show amino acid differences between the sequence
derived from the cDNA and
the published human and
guinea pig amino acid sequences.
C2b
C2a
4-w
GGC AAG CCA GGC CGU AAA AUC CAA AUC CAG CGC UCU GGU CAU CUG AAC CUC UAC CUG C-uc
(a)
Gly Lys Pro Gly Arg Lys Ile Gln Ile Gln Arg Ser Gly His Leu Asn Leu Tyr Leu ILeu
-5
10
1
(b)
Lys Ile Gln Ile Gln Ser
(c)
Lys Ile Gln Ile Gln Arg Ser Gly His Leu Asn Leu Tyr Leu Leu
X
Gly
X
Lys Asn Leu Tyr
CUA GAC UGU UCG CAG AGU GUG UCG GAA AAU GAC UUU CUC AUC UUC
(a)
Leu Asp Cys Ser Gln Ser Val Ser Glu Asn Asp Phe Leu Ile Phe
20 .30
(c)
Leu Asp Ala Ser Lys Ser Val Ser Gln Gln Asp Ile Glu
was digested with a panel of 36 restriction enzymes (EcoR I,
Pst I, BamH I, Xba I, Kpn I, Msp I, Hha I, Sac I, BstN I, Hinf
I, Bgl II, Mbo I, Nci I, Rsa I, Mlu I, Apa I, Hind III, Tthl 1 1
I, ScrF I, Mbo II, Nde I, Nae I, Sca I, EcoR V, Taq I, Sin I,
Cla I, Nar I, Xho I, Pvu II, Stu I, Xmn I, Bgl I, Ban I, Ban II,
and Bcl I) specific for different DNA sequences. This digested
DNA was analyzed by using the Southern blotting technique
and hybridized with the factor B and C2 (pC2-6) cDNA probes.
No factor B genomic polymorphisms were identified in this
panel although individuals with differing factor B protein variants
were included. In a second experiment, 23 of the enzymes were
used to analyze DNA from 20 additional unrelated individuals.
No genomic variants of factor B were identified. In contrast,
using the C2 probe to screen the initial panel of six unrelated
individuals, the DNA digested with the enzyme BamH I revealed
a polymorphism that was represented by two banding patterns,
each containing four bands (Fig. 3). Pattern I contains bands
6.9, 4.5, 3.35, and 2.3 kb and pattern II contains bands at 6.9,
6.6, 3.15, and 2.3 kb. The intensities of the bands in Fig. 3
indicate that the 6.6- and 3.15-kb bands are derived from the
4.5- and 3.35-kb bands. To date 27 unrelated individuals (54
chromosomes) have been tested for the BamH I polymorphism;
22 of these individuals were homozygous for banding pattern
I and 5 were heterozygous, i.e., show banding pattern I/IH.
Therefore, the DNA banding pattern II is represented on 10%
of the chromosomes. No individuals homozygous for the pattern
II have been identified yet; this variant would only occur at a
frequency of 1 in 100 individuals. The 22 people homozygous
for the C2 DNA pattern I include individuals with either the
C2C or the C2B protein variants. In addition, the C2 DNA
banding patterns I and I/IH have been identified in individuals
homozygous for C2 protein variant C2C. These findings demonstrate that the C2 DNA variants represent new genetic markers
distinct from the C2 protein variants. The pC2-6 cDNA probe
was digested with BamH I. No internal BamH I site was observed
(data not shown), indicating that the restriction sites detected
in genomic DNA were in flanking regions, intervening sequences,
or in coding regions not covered by the -975 bp cDNA probe.
The Mendelian inheritance ofthe C2 DNA variants is shown
in Fig. 4. Analysis of DNA from members of three generations
in this family are consistent with an autosomal codominant
0)
_
Kb
23
9.4
a)
03)
0)
oL
a.
a
E
Q
z
Kb
a
zz
am
.-
6.6w0
-
6.9
-
6.6
~41
4.4
*W
't
--:
-l.
.
-.
S.
.
3.35:
3. 1
-
2.2-
"'N
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.-
...
2.0
-
636
D. E. Woods, M. D. Edge, and H. R. Colten
A
B
Figure 3. Southern blot analysis of DNA from five unrelated individuals probed with 32P-labeled pC2-6 cDNA clone (A). Banding patterns are represented diagramatically in panel B.
Downloaded from http://www.jci.org on June 15, 2017. https://doi.org/10.1172/JCI111461
t
2
3
markers
4
4 5
6 7
Kb
9.4 _
6.6 _
4.4
-~~~~~~~
,-
4o
22
2.20
2.0
5
J
C 3 0 /it
3
1
7
/
.
Figure 4. Inheritance pattern of protein complotypes;and C2 DNA
variants. Complotypes given below each family memb3er BF, C2,
C4A, C4B-,/DNA pattern. Individual number 4 wvas repeated to
align the two Southern blots.
S C 3
fr C, 3
/
t
ISt
s C; .
s. c
mode of inheritance of the C2 genomic patterns. Ascertainment
of the protein complotypes of each family meember (Fig. 4)
reveals that in this family the C2 DNA pattern II iS associated
with the F protein variant of factor B. However, individuals
with the F variant do not always show the type I [DNA pattern
since two other unrelated individuals with this factor B type
have been identified and were homozygous for the DNA
pattern I.
used for isolation of many low abundant cDNA clones. Only
limited published amino acid sequence data for human C2 were
available (30). This sequence necessitated synthesis of an extremely complex mixture of oligonucleotides in order to include
the authentic coding sequence (Fig. 1). In the regions corresponding to amino acids at positions 3 and 5 (Glu) we constructed a mixture that contained 75% glutamine codons and
25% glutamic acid codons in order to account for possible protein
sequencing errors. The resulting oligonucleotide mixture contained 576 different sequences. A difference was found between
the amino acid at position 6 of the published sequence and that
specified by the cDNA clone (pC2-10). This difference led to a
nucleotide sequence error in the synthetic oligonucleotide probe
used to isolate the clone. Despite this error and the complexity
of the oligonucleotide mixture, a weak hybridization signal was
obtained in the initial screening of the cDNA library. The differences between the published and derived amino acid sequence
at positions 6 and 10 could be the result of errors in protein
sequencing or may represent genetic variants.
Several interesting differences between the derived human
amino acid sequence and the published sequence for guinea pig
C2a were noted. For instance, a Cys at position 18 in the human
sequence is Ala in the guinea pig, which may result in a higher
order structural difference between the guinea pig and human
C2 proteins. Such a substitution might account for the previously
recognized functional differences between guinea pig and human
C2 (33).
Overall structural and functional similarities between C2
and factor B led to speculation that these were homologous and
therefore may have arisen as a result of gene duplication. Experiments designed to detect cross hybridization even at low
stringency failed to demonstrate a high degree of homology
between these two genes. Thus, it was possible to separately
analyze genomic polymorphisms for each gene. The fact that
no genomic variants of factor B were detected plus the observation that mouse and human factor B genes display 83% nucleotide sequence homology (34) suggests that this region of the
genome has been relatively highly conserved when compared
with other genes within the MHC.
A new marker for C2 polymorphism has been identified at
the DNA level by using the Southern blotting technique. The
availability of this marker coupled with C4 variants (protein
and DNA) and C2 and factor B protein polymorphisms has
increased the potential for application of complement genetics
to the study of human diseases and for tissue typing.
Discussion
The second component of complement is synthesized in liver
(31) and mononuclear phagocytes (32) at extremely low levels,
(0.1% of total protein), hence C2-specific mRNA is of relatively
low abundance in these tissues. The availability of a human
liver cDNA library (14) containing -200,000 recombinants
allowed isolation of a C2 cDNA with the use of a synthetic
oligonucleotide mixture. Synthetic oligonucleotides have been
Acknowledgments
We thank Dr. Chester Alper for performing the protein complotyping,
Jane Ipsen and Elizabeth Davidson for technical assistance, and Ms.
Helen Hourihan for her secretarial assistance.
This work was supported by U. S. Public Health Service grants HD
17461, Al 20032, HL 32361, Al 18612, and by a grant from the Charles
H. Hood Foundation.
637
Human Complement (C2) DNA Polymorphism
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